1. Field of Invention
The present invention relates to image detection and analysis of microarray biochips, and more particularly to an apparatus and a method of retrieving and analyzing colorimetric microarray images from a microtitre plate.
2. Related Art
A biochip or microarray technology has been widely used in multi-test examination. Commercial microarray application packages for microorganism or gene tests are now available. The biochip generates a lot of information after operation. Thus it needs a biochip information detecting and analysis system that simultaneously retrieves a lot of biochip information to achieve a bulk information analysis. Most of the currently available microarray biochips are developed based on glass slides as the carriers. Nucleic acids or proteins are spotted in matrix on the glass slide as probes to detect biological characteristics in unknown samples. Glass-slide-based microarray technology usually uses a fluorescent substance as detection labels to detect any information from the glass slide. As described above, the prior art, such as a confocal laser scanner made by Axon Co. or GSI Co., is exclusively used for detection of the fluorescent signals on the glass slides.
Only one single specimen can be analyzed at a time on one single glass-slide-based microarray biochip, although multiple analytes within that specimen can be analyzed simultaneously with that biochip. This is disadvantageous to bulk on-site or clinical examination of screening purpose. The microarray biochips become popular in bulk examination because of the advantage that test of a plurality of substances to be analyzed can be performed at one time, which is also useful in various fields. The microarrays that used to be placed on the glass slides are placed in the wells on a microarray carrier such like microtitre plate. The microtitre plate is a dished container having plural (at least two) wells. Microtitre plate-based biochips are a microtitre plate with plurality of wells in bottoms of which are placed in the microarray biochips. One example of the microtitre plate is a well-known 96-well ELISA microtitre plate.
There are commercially available apparatuses used to detecting and analyzing the biochips on the microtitre plate, i.e., microarray signals of 96 samples. The detecting and analyzing apparatus are for example those made by Apibio, Pierce, Spendlove Research Foundation and High Throughput Genomics Inc. All those apparatuses cannot process colorimetric signals. Those apparatuses only detect microarray signals generated by illumination, such as fluorescent signals or chemiluminescent signals.
Colorimetric technology is less expensive than the technology using a fluorescent substance. Colorimetric technology has been applied in “flat” microarrays—one type of microarrays that microarray probes are spotted on a blotting membrane made of nylon or nitrocellulose that is soft like a paper. Data on such flat color arrays are usually retrieved by a business scanner and stored as an image file that is then analyzed by an analysis program (“Application of Enzyme Colorimetry for cDNA Microarray Detection”, Konan Peck and Yuh-Pyng Sher, in BioChip Technology, eds. Jing Cheng & Larry J. Kricka, Harwood Academic Publishers, 325-340, 2001). The membrane usually has a considerably large size such as 20 cm*10 cm, which occupies a lot of memory space. Furthermore, the membrane cannot allow bulk examination.
In terms of cost and bulk screening, there is a great demand for a colorimetric analysis of microarray biochips. FIG. 9 is a schematic view of a 96-well white scintillation micro plate as a carrier of a plural number of microarrays. A single set of microarray is placed in bottom of each well for single-sample examination. If the microtitre plate has 96 wells, then 96 samples are to be tested at one time. Meanwhile, a chromatic matrix result is generated. For example, nucleic acid probes on one set of microarrays serve to examine whether in one single food sample exist one or more of pathogenic germs such as Staphylococcus aureus, Escherichia coli, and the like. As indicated by circles in an upper portion of FIG. 10, a set of nucleic acid probes of 5×5-dot matrix is placed in each well of the 96-well microtitre plate. After reaction, only one of 8 possible colorimetric dot patterns is obtained for one well, as shown in a lower portion of FIG. 10. If all of the 96 wells undergo the reaction at the same time, it is not easy to recognize all the dot patterns of 96 wells visually, and thus the performance of bulk examination is greatly reduced. Therefore, it requires an image detection apparatus to massively process the chromatic microarray data.
However, the currently available technology retrieves the images of wells of the whole microtitre plate in sequence, which takes a long time and needs a lot of memory space for such great processing load.
The microarray biochips are mounted in the wells with sidewalls of a certain height. Such microarrays with certain thickness make the image acquisition from ordinary business scanner harder. The colorimetric signals in the microarrays on blotting membranes are located on a flat surface without any sidewall so that light uniformly distributes over and reflects by the flat surface without any concern of image brightness and uniformity. While the wells of the 96-well microtitre plate have a depth-diameter ratio of about 1.6 or more, which may cause light to be partially blocked by a part of the sidewall of the well. Non-uniform light distribution over the wells results in poor image quality. If common business scanner is used to scan the 96-well microtitre plate to retrieve the colorimetric signals in the microarrays, either the target dots cannot be focused or the single light source provided by the scanner cannot uniformly radiate over the wells to clearly retrieve the microarray images. In other words, common business scanner cannot be used in colorimetric images retrieving from deep-well microarrays.
Therefore, there is a need of a detection apparatus exclusively for detecting reflection images from microarray biochips, which can be applied in bulk examination of microarrays.